AT - DNA Alylation

Question 1

What is NER?

Answer 1

DNA damage results in bulky DNA adducts.

Recognition of the damage leads to removal of a short single-stranded DNA segment that contains the lesion.

The undamaged single-stranded DNA remains and DNA polymerase uses it as a template to synthesize a short complementary sequence.

Final ligation to complete NER and form a double stranded DNA is carried out by DNA ligase.

Note, not just damaged base, but a section of 27-30 nucleotides.

Question 2

What is the aim of chemo wrt NER?

Answer 2

Aims to overload.

Question 3

The final ligation in NER to form a double stranded DNA is done via

Answer 3

DNA Ligase

Question 4

What are the nitrogen mustards?

Answer 4

First used in WW1 as a weapon, core folds up into a 3 membered ring which is prone to nucleophile attack -> DNA is just a whole string of nucleophils.

Have two arms, can form intra or inter crosslinks.

Need to do NEH at two points so won’t work.

Nitrogen mustards needed to be softened for clinical use: eventually get to oestrogen, lots of cancers are oestrogen dependent.

Question 5

What is temozolomide?

Answer 5

Many metabolism steps are needed to get to the active compound, in short:

Has a tetrazinone ring.

Resistance is via enztme DNA alkyltransferase (AGT)

Causes mono-alkylation.

Question 6

What is mono-alkylation repaired via? [3]

Answer 6

BER
NER
06-demethylation

Question 7

How are intra-strand cross-links repaired?

Answer 7

NER

There are problems with distortion and recognition.

Question 8

How can nitrogen mustards form inter and intra strand crosslinking?

Answer 8

They are R-N(CH2-CH2-Cl)2.

The lone pair on N moves along one arm until Cl leaves, causing the ring to close up at create R-N+(CH2-CH2 ring) CH2-CH2-Cl.

The ring can act as an electrophile and be attached by nucleophillic DNA causing opening of the ring again and back to the begining structure but Cl on one arm is now DNA.

Then the other arm can do the same process and end up with two pieces of DNA linked either at locations on the same strand or a differet strand.

Question 9

How are inter-strand breaks repaired?

Answer 9

There is no repair strand/template strand for these types of breaks so we need to use DNA double strand repair (DSB repair).

Question 10

What is the use of the second strand of DNA in NER?

Answer 10

Template

Question 11

What ae DNA minor groove binders?

Answer 11

Bind to the minor groove.
Flat (planar) poly-aromatic structure, many have a natural twist.

Mitomycin C (tumour antibiotic) which binds covalently. They do not react with bases alone but are highly reactive to DNa as a whole.

Some do bind non-covalently and sit in the minor groove.

Mitomycin C binds to N2 and N7 of guanine in minor groove.

Question 12

Briefly explain how the DNA damage caused by mono-alkylating and cross-linking can be repaired.

Answer 12

Repair of Mono Alkylation: BER, NER, O6 de-methylation etc

Repair of Intra-strand cross-links: NER, problems with distortion and
recognition.

Repair of Inter-strand: No repair strand/template [DNA Double Strand (DSB)
Repair]

Question 13

Some DNA interactive ligands are able to alkylate and/or cross-link DNA,
explain the difference between mono-alkylation and inter- and intra-strand
DNA cross-linking. Illustrate your answer with examples.

Answer 13

Mono alkylation, definition and an example (Temozolomide)

Inter-strand cross-linker, definition and an example (bizelesin)

Intra-strand cross-linker, definition and an example (SJG-136/Cisplat etc)